The present invention relates to methods for the identification of an analyte in a biological medium using bioluminescence. More particularly, a method is provided for diagnosing diseases employing a solid phase methodology and a luciferase-luciferin bioluminescence generating system. Methods employing biomineralization for depositing silicon on a matrix support are also provided herein.
Bioluminescence
Luminescence is a phenomenon in which energy is specifically channeled to a molecule to produce an excited state. Return to a lower energy state is accompanied by release of a photon (hy). Luminescence includes fluorescence, phosphorescence, chemiluminescence and bioluminescence. Bioluminescence is the process by which living organisms emit light that is visible to other organisms. Luminescence may be represented as follows:
A+Bxe2x86x92X*+Y 
X*xe2x86x92X+hv, 
where X* is an electronically excited molecule and hy represents light emission upon return of X* to a lower energy state. Where the luminescence is bioluminescence, creation of the excited state derives from an enzyme catalyzed reaction. The color of the emitted light in a bioluminescent (or chemiluminescent or other luminescent) reaction is characteristic of the excited molecule, and is independent from its source of excitation and temperature.
An essential condition for bioluminescence is the use of molecular oxygen, either bound or free in the presence of a luciferase. Luciferases, are oxygenases, that act on a substrate, luciferin, in the presence of molecular oxygen and transform the substrate to an excited state. Upon return to a lower energy level, energy is released in the form of light [for reviews see, e.g., McElroy et al. (1966) in Molecular Architecture in Cell Physiology, Hayashi et al., eds., Prentice-Hall, Inc., Englewood Cliffs, N.J., pp. 63-80; Ward et al, Chapter 7 in Chemi-and Bioluminescence, Burr, ed., Marcel Dekker, Inc. NY, pp.321-358; Hastings, J. W. in (1995) Cell Physiology:Source Book, N. Sperelakis (ed.), Academic Press, pp 665-681; Luminescence, Narcosis and Life in the Deep Sea, Johnson, Vantage Press, NY, see, esp. pp. 50-56].
Though rare overall, bioluminescence is more common in marine organisms than in terrestrial organisms. Bioluminescence has developed from as many as thirty evolutionarily distinct origins and, thus, is manifested in a variety of ways so that the biochemical and physiological mechanisms responsible for bioluminescence in different organisms are distinct. Bioluminescent species span many genera and include microscopic organisms, such as bacteria [primarily marine bacteria including Vibrio species], fungi, algae and dinoflagellates, to marine organisms, including arthropods, mollusks, echinoderms, and chordates, and terrestrial organism including annelid worms and insects.
Bioluminescence, as well as other types of chemiluminescence, is used for quantitative determinations of specific substances in biology and medicine. For example, luciferase genes have been cloned and exploited as reporter genes in numerous assays, for many purposes. Since the different luciferase systems have different specific requirements, they may be used to detect and quantify a variety of substances. The majority of commercial bioluminescence applications are based on firefly [Photinus pyralis] luciferase. One of the first and still widely used assays involves the use of firefly luciferase to detect the presence of ATP. It is also used to detect and quantify other substrates or co-factors in the reaction. Any reaction that produces or utilizes NAD(H), NADP(H) or long chain aldehyde, either directly or indirectly, can be coupled to the light-emitting reaction of bacterial luciferase.
Another luciferase system that has been used commercially for analytical purposes is the Aequorin system. The purified jellyfish photoprotein, aequorin, is used to detect and quantify intracellular Ca2+ and its changes under various experimental conditions. The Aequorin photoprotein is relatively small [xcx9c20 kDa], nontoxic, and can be injected into cells in quantities adequate to detect calcium over a large concentration range [3xc3x9710xe2x88x927 to 10xe2x88x924 M].
Because of their analytical utility, many luciferases and substrates have been studied and well-characterized and are commercially available [e.g., firefly luciferase is available from Sigma, St. Louis, Mo., and Boehringer Mannheim Biochemicals, Indianapolis, Ind.; recombinantly produced firefly luciferase and other reagents based on this gene or for use with this protein are available from Promega Corporation, Madison, Wis.; the aequorin photoprotein luciferase from jellyfish and luciferase from Renilla are commercially available from Sealite Sciences, Bogart, Ga.; coelenterazine, the naturally-occurring substrate for these luciferases, is available from Molecular Probes, Eugene, Oreg.]. These luciferases and related reagents are used as reagents for diagnostics, quality control, environmental testing and other such analyses.
Chips, Arrays and Microelectronics
Microelectronics, chip arrays and other solid phase spacially addressable arrays have been been developed for use in diagnostics and other applications. At present, methods for detection of positive results are inadequate or inconvenient. There exists a need for improved, particularly more rapid detection methods.
Therefore, it is an object herein to provide detection means and methods.
A method is provided for diagnosing diseases, particularly infectious diseases, using chip methodology and a luciferase-luciferin bioluminescence generating system. A chip device for practicing the methods is also provided herein. The chip includes an integrated photodetector that detects the photons emitted by the bioluminescence generating system. The method may be practiced with any suitable chip device, including self-addressable and non-self addressable formats, that is modified as described herein for detection of generated photons by the bioluminescence generating systems. The chip device provided herein is adaptable for use in an array format for the detection and identification of infectious agents in biological specimens.
To prepare the chip, a suitable matrix for chip production is selected, the chip is fabricated by suitably derivatizing the matrix for linkage of macromolecules, and including linkage of photodiodes, photomultipliers CCD (charge coupled device) or other suitable detector, for measuring light production; attaching an appropriate macromolecule, such as a biological molecule or anti-ligand, e.g., a receptor, such as an antibody, to the chip, preferably to an assigned location thereon. Photodiodes are presently among the preferred detectors, and specified herein. It is understood, however, that other suitable detectors may be substituted therefor.
In one embodiment, the chip is made using an integrated circuit with an array, such as an X-Y array, of photodetectors. The surface of circuit is treated to render it inert to conditions of the diagnostic assays for which the chip is intended, and is adapted, such as by derivatization for linking molecules, such as antibodies. A selected antibody or panel of antibodies, such as an antibody specific for particularly bacterial antigen, is affixed to the surface of the chip above each photodetector. After contacting the chip with a test sample, the chip is contacted a second antibody linked to a component of a bioluminescence generating system, such as a luciferase or luciferin, specific for the antigen. The remaining components of the bioluminescence generating reaction are added, and, if any of the antibodies linked to a component of a bioluminescence generating system are present on the chip, light will be generated and detected by the adjacent photodetector. The photodetector is operatively linked to a computer, which is programmed with information identifying the linked antibodies, records the event, and thereby identifies antigens present in the test sample.
The chip is employed in any desired assay, such as an assay for infectious disease or antibiotic sensitivity, by, for example, linking an antibody or a panel of antibodies, to the surface, contacting the chip with a test sample of a body fluid, such as urine, blood and cerebral spinal fluid (CFS), for a sufficient time, depending upon assay format, such as to bind the a target in the sample; washing the chip and then incubating with a secondary antibody conjugated to a luciferase or an antibody:luciferase fusion protein; initiating the bioluminescent reaction; detecting light emitted at each location bound with a target through the photodiode in the chip; transferring the electronic signal from the chip to a computer for analysis.
In one embodiment, the chip is a nonself-addressable, microelectronic device for detecting photons of light emitted by light-emitting chemical reactions. The device includes a substrate, an array of loci, herein designated micro-locations, defined thereon, and an independent photodetector optically coupled to each micro-location. Each micro-location holds a separate chemical reactant that will emit photons of light when a reaction takes place thereat. Each photodetector generates a sensed signal responsive to the photons emitted at the corresponding micro-location when the reaction takes place thereat, and each photodetector is independent from the other photodetectors. The device also includes an electronic circuit that reads the sensed signal generated by each photodetector and generates output data signals therefrom. The output data signals are indicative of the light emitted at each micro-location.
In another embodiment, a microelectronic device for detecting and identifying analytes in a fluid sample using light-emitting reactions is provided. The device includes a substrate, an array of micro-locations defined thereon for receiving the fluid sample to be analyzed, a separate targeting agent attached to an attachment layer of each micro-location, and an independent photodetector optically coupled to each micro-location. Each targeting agent is, preferably, specific for binding a selected analyte that may be present in the received sample. Each photodetector generates a sensed signal responsive to photons of light emitted at the corresponding micro-location when the selected analyte bound thereto is exposed to a secondary binding agent also specific for binding the selected analyte or the targeting agent-selected analyte complex and linked to one or more components of a light-emitting reaction. The chip is then reacted with the remaining components to emit the photons when the selected analyte is present. An electronic circuit reads the sensed signal generated by each photodetector and generates output data signals therefrom that are indicative of the light emitted at each micro-location.
In yet another embodiment, a microelectronic device for detecting and identifying analytes in a biological sample using luciferase-luciferin bioluminescence is provided. The device includes a substrate, an array of micro-locations defined thereon for receiving the sample to be analyzed, a separate anti-ligand, such as a receptor antibody, attached to an attachment layer of each micro-location, and an independent photodetector optically coupled to each micro-location. Each receptor antibody is specific for binding a selected analyte that may be present in the received sample. Each photodetector generates a sensed signal responsive to bioluminescence emitted at the corresponding micro-location when the selected analyte bound to the corresponding receptor antibody is exposed to a secondary antibody also specific to the selected analyte or to the receptor antibody-selected analyte complex and linked to one or more components of a luciferase-luciferin reaction, and is then reacted with the remaining components to generate the bioluminescence when the selected analyte is present. An electronic circuit reads the sensed signal from each photodetector and generates output data signals therefrom. The output data signals are indicative of the bioluminescence emitted at each micro-location by the reaction.
In another embodiment, a method of detecting and identifying analytes in a biological sample using luciferase-luciferin bioluminescence is provided. The method includes providing a microelectronic device having a surface with an array of micro-locations defined thereon, derivatizing the surface to permit or enhance the attachment of a receptor antibody or plurality of antibodies thereto at each micro-location, and attaching a specific receptor antibody or plurality thereof to the surface at each micro-location. The selected antibody is specific for binding to a selected analyte that may be present in the sample. The method also includes applying the sample to the surface such that the selected analytes will bind to the receptor antibody attached to the surface at each micro-location, washing the sample from the surface after waiting a sufficient period of time for the selected analytes to bind with the receptor antibody at each micro-location, exposing the surface to a secondary antibody specific to bind the selected analyte already bound to the receptor antibody at each micro-location when the selected analyte is present, the secondary antibody linked to one of a luciferase and a luciferin, and initiating the reaction by applying the other of the luciferase and luciferin to the surface. The method also includes detecting photons of light emitted by the reaction using a photodetector optically coupled to each micro-location, each photodetector generating a sensed signal representative of the bio-luminescent activity thereat, reading the sensed signal from each photodetector and generating output data signals therefrom indicative of the bioluminescence emitted at each micro-location by the reaction.
In a further embodiment, a system for detecting and identifying analytes in a biological sample using luciferase-luciferin bioluminescence is provided. The system includes: a microelectronic device including an array of micro-locations for receiving the sample; a separate receptor antibody attached to an attachment layer of each micro-location, each receptor antibody is specific for a selected analyte that may be present in the received sample; a photodetector that generates a sensed signal responsive to bioluminescence emitted at the corresponding micro-location when the selected analyte bound to the corresponding receptor antibody is exposed to a secondary antibody also specific to the selected analyte and linked to one of a luciferase and a luciferin, and is then reacted with the other of the luciferase and luciferin to generate the bioluminescence when the selected analyte is present, and an electronic circuit which reads the sensed signal from each photodetector and generates output data signals therefrom indicative of the bioluminescence emitted at each micro-location by the reaction. The system includes a processing instrument including an input interface circuit for receiving the output data signals indicative of the bioluminescence emitted at each micro-location, a memory circuit for storing a data acquisition array having a location associated with each micro-location, an output device for generating visible indicia in response to an output device signal and a processing circuit. The processing circuit reads the output data signals received by the input interface circuit, correlates these signals with the corresponding micro-locations, integrates the correlated output data signals for a desired time period by accumulating them in the data acquisition array, and generates the output device signal which, when applied to the output device, causes the output device to generate visible indicia related to the presence of the selected analytes.
In other embodiments, the chip is self-addressable. When using self-addressable chips in the method, presently preferred are those adaptable to microelectronic self addressable, self-assembling chips and systems, such as those described in International PCT application Nos. WO 95/12808; WO 96/01836 and WO 96/07917 and also in arrays, such as those described in U.S. Pat. No. 5,451,683, which are each herein incorporated by reference. The self-addressable chips are such that each individual well may be addressed one at a time in the presence of the rest by changing the charge at a single microlocation and then sending the analytes or reagents via free flow electrophoresis throughout, but assembly occurs only at that location after the chip has been assembled. These devices are modified for use in the methods herein by replacing the disclosed detection means with the luciferase/luciferin systems.
In another embodiment provided herein, electrodes, an anode and cathode, are located at the bottom and top of each well, respectively, to allow for the delivery of analytes and reagents by free flow electrophoresis. The antibodies are attached to each location on a MYLAR (oriented polyethylene terephthalate) layer prior to assembling the chip (using, for example, a dot matrix printer). Thus, it is nonself-addressable in that is has a plurality of individual wells each containing a photodiode incorporated into the semiconductor layer at the bottom of each well.
In practice, for example, specific anti-ligands, e.g., antibodies, may be attached directly to the matrix of the chip or to a middle reflective support matrix, such as heat stable MYLAR, positioned in the center of each well. The sample is contacted with the chip, washed and a plurality of secondary antibody-luciferase conjugates or protein fusions are added. The wells are washed and the remaining components of the bioluminescent reaction are added to initiate the reaction. Light produced in a well is detected by the photodiode, photomultiplier, CCD (charge coupled device) or other suitable detector in the semiconductor layer and the signal is relayed to a processing unit, typically a computer. The processing unit displays the well or wells that are positive. Each well corresponds to a particular ligand, thereby permitting identification of the infectious agents. All steps may be automated.
The design, fabrication, and uses of nonself-addressable and programmable, self-addressable and self-assembling microelectronic systems and devices which actively carry out controlled multi-step and multiplex reactions in microscopic formats for detecting the electromagnetic emissions of a bioluminescent reaction are provided herein. The reactions include, but are not limited to, most molecular biological procedures, such as nucleic acid and protein nucleic acid hybridizations, antibody/antigen reactions, and related clinical diagnostics.
The resulting chips, which includes a silicon matrix and photodiodes or other light detecting means, are provided. The silicon may be deposited using enzymatic deposition, similar to the enzymatic deposition by radiolarains and diatoms. Also provided are chips in which the absorption of silica or derivatives thereof is advantageously employed as a detection means. Such silica has an absorption maxima at about 705 nm, which is the wavelength emitted by Aristostomias bioluminescence generating system. Enzymatic methods for depositing silicon on the surface of a matrix are also provided herein.
Also provided herein is a synthetic synapse. A suitable enzyme, particularly, acetylcholine esterase is fused to a luciferase, such as by recombinant expression. The luciferase is either in an inactive or active conformation. Suitable mutations in either protein may be selected to insure that luciferase can undergo appropriate conformational changes as described herein. The resulting fusion is attached to a chip, such as a chip provided herein. Upon binding of the ligand to the enzyme, such as the binding of acetylcholine to the esterase, the linked luciferase is, if previously inactive, is activated by the binding, or if previously active, is inactivated by the binding. In the presence of the remaining components of a bioluminescence generating system, light is produced (or is quenched), which change is detected by the photodiodes associated with the chip. This detection generates an signal that is processed, such as by a computer, and is transmitted by appropriate means, such as fiber, to an electrode, which is attached to any desired device or effector, particularly a muscle. Upon receipt of the signal, work, such as a muscle twitch, occurs.